Early embryo development, defined as the period starting with oocyte maturation and fertilization and ending with blastocyst formation also known as pre-implantation, is a period in which many embryos die or cease development. This appears to be significantly higher when embryos are produced by in vitro fertilization or cloning (in animals) and/or after cryopreservation. Several important events in early embryo development include oocyte maturation and sperm capacitation, fertilization, cleavage, compaction, and blastocyst formation. In the in vivo condition these events occur in the female reproductive tract, which provides an optimal environment for embryo development.
With the advent of Assisted Reproductive Technology a revolution happened in reproduction biology and biotechnology, which resulted in the production “test tube” embryos in different species. With respect to humans, infertility treatment is widely implemented given the abundance of advanced reproductive technologies available. Currently, the success rate of flushing a woman for multiple oocytes, followed by in vitro fertilization, embryo transfer and pregnancy is quiet low (<25%). Most women have to go through multiple cycles of hormone stimulation to obtain multiple embryos. Hormone stimulation is a significant risk with possible severe negative effects. Consequently, any aspect of in vitro embryo production (IVP) that can increase the resiliency and success of embryo survival will reduce the number of embryos needed to be collected and the number of times a woman is super ovulated in order to obtain oocytes to produce embryos by in vitro fertilization (IVF) for embryo transfer.
Further, with the development of cryopreservation, eggs and/or fertilized eggs (embryos) may be stored for future use, such as embryo transfer. With respect to humans, this may allow women to store young normally ovulated eggs and/or fertilized eggs (embryos) obtained during the prime reproductive years, and use them when they are older. Current methods of cryopreservation remain problematic since frozen-thawed embryos lack viability and are prone to apoptosis, which limits their utility in embryo transfer. Thus, any aspect of cryopreservation that improves the survival of eggs or embryos post cryopreservation is tremendously beneficial.
One of the most important parts of in vitro embryo production is culture media and its composition for the various stages of early embryo development. For the past four decades researchers in this field have attempted to optimize the usefulness of in vitro media, including media for in vitro maturation (IVM) of oocytes, media for in vitro fertilization (IVF) of oocytes with sperm, and media for in vitro culture (IVC) of embryos. However in vitro embryo development and survival remains problematic. Each period in early embryo development represents different stages which have distinct growth factor requirements. In vivo, there are tremendous autocrine, paracrine and endocrine factors which are integrated and act during the different stages of early embryo development.
An example of these factors are the thyroid hormones, produced and secreted by the thyroid gland in response to stimulation by thyroid stimulating hormone (TSH), which is released by the pituitary gland. In vivo, thyroid hormones are mainly expressed in two forms, thyroxine (T4) and triiodothyronine (T3) and at a serum concentration ratio of approximately 20:1, respectively. In blood, most of this thyroid hormone is bound to carrier protein molecules (thyroxine-binding globulin, transthyretin, or albumin). In blood, unbound hormone is called free thyroid hormone which is biologically more active than bound thyroid hormone. Free T3 (fT3) is three to four times more potent than free T4 (fT4) and is created as needed within tissues using deiodinases (5′-iodinase) to convert T4 to T3. Thyroid hormones play an important role in vertebrate growth, differentiation and metabolism.
For example, one study indicated that infertile immature spontaneously hypothyroid RDW female rats had significantly more ovulated eggs and improved follicular development following treatment with T4 and equine chorionic gonadotropin (eCG) (Sato E et al. 2001). Treatment of bovine granulosa cells with T3 and T4 caused an increase in net estrogen production (L. J Spicer 2001). T3 synergizes with follicle-stimulating hormone (FSH) to induce differentiation of granulosa cells in porcine follicles (Maruo et al. 1987).
The use of thyroid hormone for initial stages of in vitro oocyte maturation is disclosed in U.S. Pat. No. 4,987,080 (issued in 1991). Specifically, this patent discloses incubation of oocytes in culture media containing one or more thyroid hormones for the growth and development of small and medium oocytes into large oocytes. However, this patent suggests and discloses the use of a different culture media that does not include thyroid hormones for subsequent stages of development, including ova maturation, in vitro fertilization, early cleavage of the embryo, and growth of the embryo to the blastocyst stage. The culture media disclosed for use in these steps is described as having low nutrients and a high energy source, and may include bovine serum albumin.